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骨髓巨噬细胞M2亚型共培养后的骨髓间充质干细胞移植治疗肝硬化大鼠模型的效果分析

郑欣瑞 许燕楠 王丹阳 邢飞飞 宗梦瑶 张士豪 战俊邑 刘伟 陈高峰 陈佳美 刘平 慕永平

引用本文:
Citation:

骨髓巨噬细胞M2亚型共培养后的骨髓间充质干细胞移植治疗肝硬化大鼠模型的效果分析

DOI: 10.12449/JCH240117
基金项目: 

国家自然科学基金面上项目 (81874390)

上海市科委自然科学基金面上项目 (21ZR1464100)

上海市科委2022年度“科技创新行动计划”生物医药科技支撑专项 (22S11901700)

上海市临床重点专科建设项目 (shslczdzk01201)

伦理学声明:本研究方案于2021年11月15日经由上海中医药大学动物研究委员会批准,批号:PZSHUTCM211115023,符合实验室动物管理与使用准则。
利益冲突声明:本文不存在任何利益冲突。
作者贡献声明:郑欣瑞、许燕楠负责实验实施,收集数据,资料分析,论文撰写;王丹阳、邢飞飞、张士豪、宗梦瑶、战俊邑参与实验研究;陈高峰负责病理;刘伟、陈佳美、刘平参与实验设计;慕永平负责课题设计,提供写作思路,指导论文撰写并最后定稿。郑欣瑞、许燕楠对本文贡献等同,同为第一作者。
详细信息
    通信作者:

    慕永平, ypmu8888@126.com (ORCID: 0000-0002-6808-8243)

Therapeutic effect of transplantation of bone marrow mesenchymal stem cells co-cultured with bone marrow M2 macrophages on a rat model of liver cirrhosis

Research funding: 

National Natural Science Foundation of China (81874390);

Shanghai Natural Science Foundation (21ZR1464100);

Special Project for Biomedical Science and Technology Support of the “Science and Technology Innovation Action Plan” of Shanghai Science and Technology Commission in 2022 (22S11901700);

Shanghai Key Specialty of Traditional Chinese Clinical Medicine (shslczdzk01201)

More Information
    Corresponding author: MU Yongping, ypmu8888@126.com (ORCID: 0000-0002-6808-8243)
  • 摘要:   目的  探讨骨髓巨噬细胞M2亚型(M2-BMDM)共培养后的骨髓间充质干细胞(BMSCM2)移植对四氯化碳/2-乙酰氨基芴(CCl4/2-AAF)诱导肝硬化大鼠模型进展的影响。  方法  分离大鼠BMDM并极化为M2表型;分离大鼠BMSC,培养至第3代时与M2-BMDM共培养后获取BMSCM2。CCl4皮下注射6周建立大鼠肝硬化模型。将模型大鼠随机分为模型组(M组)、BMSC组、BMSCM2组,同时设有正常组(N组),每组6只。第7周开始,模型大鼠于CCl4皮下注射的同时予以2-AAF灌胃,分组干预,10周末取材,观察肝功能、肝组织病理、肝组织羟脯氨酸(Hyp)含量,以及肝星状细胞、肝祖细胞、胆管细胞、肝细胞标志物的变化情况。计量资料多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。  结果  与N组比较,M组大鼠血清ALT、AST活性均显著升高(P值均<0.01);与M组比较,BMSC组和BMSCM2组大鼠ALT、AST均显著降低(P值均<0.01),且BMSCM2组显著优于BMSC组(P值均<0.05)。与N组比较,M组大鼠肝脏Hyp含量、α-SMA mRNA及蛋白表达均显著升高(P值均<0.01);与M组比较,BMSC组和BMSCM2组Hyp含量、α-SMA表达均显著降低(P值均<0.05),且BMSCM2组α-SMA水平显著低于BMSC组(P<0.01)。与N组比较,M组大鼠肝祖细胞标志物EpCam、Sox9以及胆管细胞标志物CK7、CK19 mRNA表达均显著增加(P值均<0.01),肝细胞标志物HNF-4α和Alb表达均显著降低(P值均<0.01);与M组比较,BMSC组和BMSCM2组EpCam、Sox9、CK7和CK19 mRNA表达均显著降低(P值均<0.05),HNF-4α和Alb mRNA表达均显著增加(P值均<0.05);且与BMSC组比较,BMSCM2组EpCam和CK19 mRNA表达均显著降低(P值均<0.05),而HNF-4α mRNA表达显著增加(P<0.05)。  结论  M2-BMDM可提高BMSC对CCl4/2-AAF诱导大鼠肝硬化的治疗效应,为进一步提高BMSC治疗肝硬化的作用提供了新思路。

     

  • 图  1  BMDM和BMSC鉴定

    注: a,BMSC镜下形态学观察(×100);b,BMSC成骨诱导(茜素红染色,×100);c,BMSC成脂诱导(油红O染色,×100);d,M2-BMDM流式细胞鉴定;e,BMSC细胞周期检测;f,BMSC流式细胞鉴定。

    Figure  1.  BMDM and BMSC identification

    图  2  BMSCM2抑制肝脏炎症反应

    注: a,HE染色(×200);b,CD68免疫组化染色(×200);c,血清ALT、AST活性;d,肝组织TNF-α、TGF-β1及CD68 mRNA表达水平;e,肝组织CD68免疫印迹;f,肝组织CD68免疫印迹灰度积分比值。

    Figure  2.  BMSCM2 inhibits hepatic inflammatory response

    图  3  天狼星红染色和α-SMA免疫组化染色结果(×200)

    Figure  3.  Sirius red staining and α-SMA immunohistochemical staining results(×200)

    图  4  BMSCM2抑制肝硬化进展

    注: a,肝组织Hyp含量;b,肝组织α-SMA mRNA表达水平;c,肝组织α-SMA免疫印迹;d,肝组织α-SMA免疫印迹灰度积分比值。

    Figure  4.  BMSCM2 inhibits the progression of liver cirrhosis

    图  5  EpCam和Sox9免疫组化染色结果(×200)

    Figure  5.  Immunohistochemical staining results of EpCam and Sox9 (×200)

    图  6  BMSCM2抑制肝祖细胞增殖

    Figure  6.  BMSCM2 inhibits hepatic progenitor cell proliferation

    图  7  CK7和CK19免疫组化染色结果(×200)

    Figure  7.  Immunohistochemical staining results of CK7 and CK19 (×200)

    图  8  BMSCM2抑制胆管反应

    Figure  8.  BMSCM2 inhibits bile duct reactions

    图  9  HNF-4α和Alb免疫组化染色结果(×200)

    Figure  9.  Immunohistochemical staining results of HNF-4α and Alb (×200)

    图  10  BMSCM2 促进肝细胞增殖

    Figure  10.  BMSCM2 promotes hepatocyte proliferation

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  • 收稿日期:  2023-04-24
  • 录用日期:  2023-05-15
  • 出版日期:  2024-01-23
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